Process for the manufacture of highly active substances from the posterior lobe of the hypophysis



Patented Jan. 12, 1943 w p I 2,308,287

UNITED STATES PATENT OFFICE PROCESS FOR THE MANUFACTURE HIGHLY ACTIVE SUBSTANCES FROM THE POSTERIOR LOBE OF THE HYP'OPHYSIS Karl Junkmann, Berlin, Germany, assignor .to I Schering Corporation, Bloomfield, N. 1., acorporation of New Jersey No Drawing. Application December 12,"1998,-Se-

rial No.-245,229. In Germany December 14,

1937 I 1 Claim. (01.167-74) This invention relates to highly active subthe hormones contained'in the starting'imaterial stances from the posterior lobe of the hypophysis on uterus and on blood pressure.

and a process of making the same. The following example illustrates the inven- The production of active substances from the tion: posterior lobe of the hypophysis is already known 5 3 Example from the literature. Thus Trendelenburg, Die Hormone, volume I, pages 133-173, published 1929, in Berlin, discloses particularly on page, 141, that the posterior lobe of the hypophysis contains physiologically active principles that increase the blood pressure and that directly stimulate muscular tissue such as the uterus. Fiihner Zeitschrift iiir-die gesamte experimentelle Medizin vol. I, page 399/400-1913, -Therapeutische Halbmonatshefte 34/1920, page 437, and also other authors employed for the precipi- I tation of the active substances from aqueous extracts of posterior lobe phosphotungstic acid. It is true that thereby an active precipitate was obtained but it was not possible to separate or isolate in good yield from such a precipitate the individual active substances, compare Abderhalden Handbuch der biologischen Arbeitsme- 50 grams of hypophysis posterior lobe dry powder with 80,000 international units are boiled for 5 minutes with 2.5 litres of weak acetic acid (0.25%), filtered and the precipitate washed with water until the filtrate amounts to about 2.5 litres. After cooling of the filtrate to 0 C. it is precipitated with the calculated quantity of phosphotungstic acid (80 cos. of 10% acid dissolved in 10% sulphuric acid), after 1 hour's standing the whole is centrifuged in a cold chamber and the precipitate washed with a small quantity of dilute (about 3%) phosphotungstic acid solution. The precipitate is then suspended in 400 cos. of methyl alcohol cooled to 10" C. and treated with likewise correspondingly cooled methyl alcoholic saturated baryta solution to a distinctly alkaline reaction (phenol phthaleln red), whereby about. 100 ccs. are employed. thden pm 31mg: ms After 30 minutes the whole is centrifuged in a further known (German Patent No. 264,119) 25 cool chamber, the solution nted, the precipithat P Prevmusly Purmed: albumeP'me tate washed with a little methyl alcohol and the pophysls GXtYaCtS precipltation heavy combined methyl alcoholic solutions exactly neumetal Partlcularly mercury salts tralised for quantitative precipitation of the baactlve constituents can be precipitated and after 20 mun with dilute sulphuric acids in which case remmal metal 9 be tamed in suitably the completion of the precipitation is formq m to thls process mwever, effected by the addition of a small quantity of a small Yield 15 f solid sodium sulphate. vThe barium sulphate In accordance Wlth the e inventmn precipitate is filtered of! and the filtrate if dehighly active Preparation. is Obtained in solid sired after weak acidification with acetic acid". form and practically quantitative yield when the evaporated to dryness in a good vacuum; The active precipitate Obtained by precipitation'with residue is dissolved in 15 cos. of glacial acetic; acid precipitating agents is freed from the preacid. filtered for removal of insoluble salts and cipitating agent by treating the precipitate with the filtrate precipitated with ether. The fil-f a o u n 01 Suspension o e in aqueOus 40 tered'precipitate in-the completely dryconditlon organic solvents for the active substances at a is dissolved i 10 of glacial I a eti p a e e ow sui a below C- (98%), the solution filtered clear, the {filtrate rt l r y good results are Ob n d wh treated with 40 ccs.'of ether and the precipitateh precipitation of he esubstances is producedwashed with, oth r .and dri d. .Yieldi. car ied out by means of p sp -tunssti acid. 5 500-250 mg. of awhite powder containingfiopoof tunsst c c d. mo ybd ac p pho y i. international units of each of the hormonalsub 1' acid and others atlow temperature and the acid stances acting on. the uterus andon blood presemployed for the precipitation is removed from sure.- 1mg; accordingly cont'ains240-120 interthe precipitate with aqueous alcoholic alkaline national units, of thespecified main activities. earth hydroxide solution or suspension, for ex- Th preparation t t m can be ample, with methyl alcoholic baryta solution at ployed directly as pharmaceutical product, howtemperatures below 0 C. suitably below -l0 C. ever, also the substance raising the blood. pres The end product is obtained in this manner sure contained therein canbeseparated-withou in a very good yield and if desired purified by difiiculty and employed separately. 1'. I

several reprecipitations exhibits the effects of Of course, many changes and variations in the reaction conditions and the like may be employed by those skilled in the art in accordance with the principles set forth herein and inthe claim annexed hereto.

What I claim is:

In a process for separating physiologically highly active substances i'rom'a precipitate resulting from the action upon a liquid extract of the posterior lobe of the hypophysis of an acid selected from the group consisting of phosphotungstic acid, tungstic acid, molybdic acid, and phosphomolybdic acid, the steps comprising suspending said precipitate in a chilled, water-miscible organic solvent for the active substances, treating the suspension with a saturated solution oi barium hydroxide in aqueous methyl alcohol cooled to a temperature no higher than about -10 C. to liberate the physiologically active substances and iorm an insoluble barium salt 01' the acid precipitating agent, separating the solution of active substances from the insoluble barium salt, treating the solution of active substances with precisely enough dilute suliuric acid to neutra'lize and precipitate any barium hydroxide present as barium sulfate, removing the barium sulfate, and evaporating the solution to dryness, and subjecting the resulting residue containing the physiologically active substances to at least one purification treatment consisting of dissolving the residue in an organic acid, filtering, precipitating the active substances from the filtrate, separating and drying the precipitate, and finally isolating, from the mixture of active substances in the precipitate a principle having as its chief property the raising of the human blood pressure and a principle having as its chief property a direct stimulating action on the muscular tissue of the uterus. 

